Multiplex data collection and analysis in bioanalyte detection

ABSTRACT

Method and device to collect multiplex data simultaneously in analyte detection and analyze the data by experimentally trained software (machine-learning) is disclosed. Various ways (magnetic particles and microcoils) are disclosed to collect multiple reporter (tag) signals. Multiplex detection can increase the biomolecule analysis efficiency by using small sample size and saving assay reagents and time. Machine learning and data analysis schemes are also disclosed. Multiple affinity binding partners, each labeled by a unique reporter, are contacted with a sample and a single spectrum is taken to detect multiple reporter signals. The spectrum is deconvoluted by experimentally trained software to identify multiple analytes.

RELATED APPLICATIONS

This application is related to U.S. patent application Ser. No.10/748,336, filed Dec. 29, 2003, entitled “Composite Organic-InorganicNanoparticles (COIN) as SERS Tags for Analyte Detection,” U.S. patentapplication Ser. No. 10/916,710, filed Aug. 11, 2004, entitled“Multiplex Detection of Analytes in Fluid Systems,” U.S. patentapplication Ser. No. 10/927,996, filed Aug. 26, 2004, entitled“Biomolecule Analysis Using Raman Surface Scanning”, and U.S. patentapplication Ser. No. 11/027,470, filed Dec. 30, 2004, entitled“Biomolecule Analysis Using Raman Surface Scanning.”

FIELD OF INVENTION

The embodiments of the invention relate to methods and devices forcomplex data collection and analysis in multiplexed biomoleculedetection. The invention transcends several scientific disciplines suchas polymer chemistry, biochemistry, molecular biology, medicine andmedical diagnostics.

BACKGROUND

The molecular-level origins of disease are being elucidated at a rapidpace, potentially ushering in a new era of personalized medicine inwhich a specific course of therapy is developed for each patient. Tofully exploit this expanding knowledge of disease phenotype, new methodsfor detecting multiple biomolecules (e.g., DNA and proteins)simultaneously are required. The multiplex biomolecule detection methodsmust be rapid, sensitive, highly parallel, and ideally capable ofdiagnosing cellular phenotype in vivo.

Some biomolecule detection methods have been developed based upon theunique electrochemical and photoelectrochemical properties of metalparticles. In one assay method, gold nanoparticles (10 nm diameter) aretagged with ssDNA probe strands and a photoactive dye molecule. A metalelectrode of a microarray chip (also called gene chip) is also modifiedwith ssDNA probe strands. If a target (the analyte or bioanalyte) mRNAor ssDNA is complementary to the probe on the particle and thesubstrate, hybridization will occur which brings the particle in contactwith the electrode. A laser is then rastered across the surface. Whenthe laser addresses a spot in which nanoparticles are bound, the dyemolecule is electronically excited, and the excited electron is injectedinto the electrode. The electron is collected as a current, signifyingthe presence of a particular DNA analyte.

Synthesis of a functionalized electrode having polymer arrays on anelectrode of a microarray chip is known. Examples of such polymer arraysinclude nucleic acid arrays, peptide arrays, and carbohydrate arrays.

One method of preparing functionalized electrodes of polymer arrays onmicroarray chips involves photolithographic techniques usingphotocleavable protecting groups. Briefly, the method includes attachingphotoreactive groups to the surface of a substrate, exposing selectedregions of the substrate to light to activate those regions, attaching amonomer with a photoremovable group to the activated regions, andrepeating the steps of activation and attachment until macromolecules ofa length and sequence are synthesized.

Additional methods and techniques applicable to prepare a functionalizedelectrode include electrochemical synthesis. One example includesproviding a porous substrate with an electrode therein, placing amolecule having a protected chemical group in proximity of the poroussubstrate, placing a buffering solution in contact with the electrodeand the porous substrate to prevent electrochemically generated reagentsfrom leaving the locality of the electrode (the use of confinementelectrodes to prevent reagents from diffusing away have also beendescribed), applying a potential to the electrode to generateelectrochemical reagents capable of deprotecting the protected chemicalfunctional group of the molecule, attaching the deprotected chemicalfunctional group to the porous substrate or a molecule on the substrate,and repeating the above steps until polymers of a length and sequenceare synthesized.

The biomolecules on microarray chip typically are detected throughoptical readout of fluorescent labels attached to a target molecule thatis specifically attached or hybridized to a probe molecule. Theseoptical methods are difficult to implement and miniaturize because theyrely on the use of optical labels and require large or expensiveinstrumentation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the methods and devices to create sub-set ofbinding complexes.

FIG. 2 shows the sample preparation method for a micro-fluid channelbased multiplex analyzer system.

FIG. 3 shows the detection methodology for a micro-fluid channel basedmultiplexed analyzer systems.

FIG. 4 shows the sample preparation method for a micro-array basedmultiplexed analyzer systems.

FIG. 5 shows the detection methodology for a micro-array basedmultiplexed analyzer systems.

FIG. 6 shows a schematic of the magnetic COIN as reporter and analytecarrier.

FIG. 7 shows a schematic of the microcoil for concentratingmagnetic-COIN sandwich complexes.

FIG. 8 shows a schematic of a system for machine learning and analytequantification.

FIG. 9 shows Raman spectra of multiplexed COIN mixtures.

DETAILED DESCRIPTION

A biological sample often contains many thousands or even more types ofbiomolecules and clinical diagnosis needs to measure multiple analytesfor disease confirmation. Currently, each analyte is measuredseparately, which requires multiple samples from a patient. Theprocedure is time consuming and labor intensive. The embodiments of theinvention allow for multiple analyte detection from a single sample anda single test, which could be of great interest to clinical diagnosis,and biomedical research as well.

Analytes include nucleic acids (DNA and RNA), which can formdouble-stranded molecules by hybridization, that is, complementary basepairing. The specificity of nucleic acid hybridization is such that thedetection of molecular and/or nanomaterials binding events can be donethrough electrical readout of polarization changes caused by theinteraction of charged target molecules (DNA, RNA, proteins, forexample.) and chemically modified nanomaterials (carbon nanotubes,nanowires, nanoparticles functionalized with DNA, for example) withcomplementary molecular probes (DNA, RNA, anti-body, for example)attached to the electrodes (such as Au, Pt, for example). Thisspecificity of complementary base pairing also allows thousands ofhybridization to be carried out simultaneously in the same experiment ona DNA chip (also called a DNA array).

Molecular probes are immobilized on the surface of individuallyaddressable electrode arrays through the surface functionalizationtechniques. Electrodes allow polarization changes to be electricallydetected. The polymer arrays of the embodiment of the invention could bea DNA array (collections of DNA probes on a shared base) comprising adense grid of spots (often called elements or pads) arranged on aminiature support. Each spot could represent a different gene.

The probe in a DNA chip is usually hybridized with a complex RNA or cDNAtarget generated by making DNA copies of a complex mixture of RNAmolecules derived from a particular cell type (source). The compositionof such a target reflects the level of individual RNA molecules in thesource. The intensities of the signals resulting from the binding eventsfrom the DNA spots of the DNA chip after hybridization between the probeand the target represent the relative expression levels of the genes ofthe source.

The DNA chip could be used for differential gene expression betweensamples (e.g., healthy tissue versus diseased tissue) to search forvarious specific genes (e.g., connected with an infectious agent) or ingene polymorphism and expression analysis. Particularly, the DNA chipcould be used to investigate expression of various genes connected withvarious diseases in order to find causes of these diseases and to enableaccurate treatments.

Using embodiments of the invention, one could find a specific segment ofa nucleic acid of a gene, i.e., find a site with a particular order ofbases in the examined gene. This detection could be performed by using adiagnostic polynucleotide made up of short synthetically assembledsingle-chained complementary polynucleotide—a chain of bases organizedin a mirror order to which the specific segment of the nucleic acidwould attach (hybridize) via A-T or G-C bonds.

The practice of the embodiments of the invention may employ, unlessotherwise indicated, conventional techniques of organic chemistry,polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “an array” may include a plurality ofarrays unless the context clearly dictates otherwise.

An “array” is an intentionally created collection of molecules which canbe prepared either synthetically or biosynthetically. The molecules inthe array can be identical or different from each other. The array canassume a variety of formats, e.g., libraries of soluble molecules;libraries of compounds tethered to resin beads, silica chips, or othersolid supports. The array could either be a macroarray or a microarray,depending on the size of the sample spots on the array. A macroarraygenerally contains sample spot sizes of about 300 microns or larger andcan be easily imaged by gel and blot scanners. A microarray wouldgenerally contain spot sizes of less than 300 microns.

“Solid support,” “support,” and “substrate” refer to a material or groupof materials having a rigid or semi-rigid surface or surfaces. In someaspects, at least one surface of the solid support will be substantiallyflat, although in some aspects it may be desirable to physicallyseparate synthesis regions for different molecules with, for example,wells, raised regions, pins, etched trenches, or the like. In certainaspects, the solid support(s) will take the form of beads, resins, gels,microspheres, or other geometric configurations.

The term “probe” or “probe molecule” refers to a molecule attached tothe substrate of the array, which is typically cDNA or pre-synthesizedpolynucleotide deposited on the array. Probes molecules are biomoleculescapable of undergoing binding or molecular recognition events withtarget molecules. (In some references, the terms “target” and “probe”are defined opposite to the definitions provided here.) Thepolynucleotide probes require only the sequence information of genes,and thereby can exploit the genome sequences of an organism. In cDNAarrays, there could be cross-hybridization due to sequence homologiesamong members of a gene family. Polynucleotide arrays can bespecifically designed to differentiate between highly homologous membersof a gene family as well as spliced forms of the same gene(exon-specific). Polynucleotide arrays of the embodiment of thisinvention could also be designed to allow detection of mutations andsingle nucleotide polymorphism.

The term “target” or “target molecule” refers to a small molecule,biomolecule, or nanomaterial such as but not necessarily limited to asmall molecule that is biologically active, nucleic acids and theirsequences, peptides and polypeptides, as well as nanostructure materialschemically modified with biomolecules or small molecules capable ofbinding to molecular probes such as chemically modified carbonnanotubes, carbon nanotube bundles, nanowires and nanoparticles. Thetarget molecule may be fluorescently labeled DNA or RNA.

The terms “die,” “polymer array chip,” “DNA array,” “array chip,” “DNAarray chip,” “bio-chip” or “chip” are used interchangeably and refer toa collection of a large number of probes arranged on a shared substratewhich could be a portion of a silicon wafer, a nylon strip or a glassslide.

The term “molecule” generally refers to a chemical made up of two ormore atoms and includes a macromolecule, biomolecule or polymer asdescribed herein. However, arrays comprising single molecules, asopposed to macromolecules or polymers, are also within the scope of theembodiments of the invention. The term “biomolecule” refers to anyorganic molecule that is part of a living organism. A “complex of abiomolecule” refers to a structure made up of two or more types ofbiomolecules. Examples of a complex of biomolecule include a cell orviral particles. A cell can include bacteria, fungi, animal mammaliancell, for example.

“Predefined region,” “spot” “binding area” or “pad” refers to alocalized area on a solid support which is, was, or is intended to beused for the formation of a selected molecule and is otherwise referredto herein in the alternative as a “selected” region. The predefinedregion may have any convenient shape, e.g., circular, rectangular,elliptical, wedge-shaped, etc. For the sake of brevity herein,“predefined regions” are sometimes referred to simply as “regions” or“spots.” In some embodiments, a predefined region and, therefore, thearea upon which each distinct molecule is synthesized is smaller thanabout 1 cm² or less than 1 mm², and still more preferably less than 0.5mm². In most preferred embodiments the regions have an area less thanabout 10,000 μm² or, more preferably, less than 100 μm². Additionally,multiple copies of the polymer will typically be synthesized within anypreselected region. The number of copies can be in the thousands to themillions. More preferably, a die of a wafer contains at least 400 spotsin, for example, an at least 20×20 matrix. Even more preferably, the diecontains at least 2048 spots in, for example, an at least 64×32 matrix,and still more preferably, the die contains at least 204,800 spots in,for example, an at least 640×320 array. A spot could contain anelectrode to generate an electrochemical reagent, a working electrode tosynthesize a polymer and a confinement electrode to confine thegenerated electrochemical reagent. The electrode to generate theelectrochemical reagent could be of any shape, including, for example,circular, flat disk shaped and hemisphere shaped.

A “microcoil” refers to a localized microelectromagnet on or in a solidsupport which is, was, or is intended to be used for the formation of aselected molecule under the influence of magnetic field. Integratedmicrocoils in an array may have any convenient shape, e.g., circular,rectangular, elliptical, wedge-shaped, etc. In some embodiments of theinvention, the microcoil could be smaller than about 1 cm² or less than1 mm², and still more preferably less than 0.5 mm². In most preferredembodiments the microcoil could have an area less than about 10,000 μm²or, more preferably, less than 100 μm². For independent magnetic fieldcontrol, each microcoil is connected to its own on-chip current source.The operating principle of the microcoil array for cell manipulation isto create and move magnetic field peaks by modulating currents in themicrocoils. For instance, by activating only one microcoil in the array,a magnetic bead suspended in fluid will be attracted to the field peakat the center of the microcoil on the surface of the IC having themicrocoil. Subsequently, by turning off the microcoil while activatingan adjacent one, the magnetic field peak is moved to the center of theadjacent microcoil, transporting the magnetic bead to the new peaklocation. The spatial resolution of the manipulation is determined bythe spacing between two neighboring coils. For precise spatial controlof individual magnetic beads, the microcoil could be carefully designedto generate a single magnetic field peak on the chip surface. Note thatwhile the microcoil generally produces a single magnetic peak on thechip surface, multiple magnetic peaks can exist below the surface.

An “electrode” is a body or a location at which an electrochemicalreaction occurs. The term “electrochemical” refers to an interaction orinterconversion of electric and chemical phenomena. A “functionalizedelectrode” is an electrode of a microchip array having a probe moleculethat has a specific chemical affinity to a target molecule. An“unfunctionalized electrode” is an electrode of a microchip array havingno probe molecule or having a probe molecule that has no specificchemical affinity to a target molecule.

The electrodes used in embodiments of the invention may be composed of,but are not limited to, metals such as iridium and/or platinum, andother metals, such as, palladium, gold, silver, copper, mercury, nickel,zinc, titanium, tungsten, aluminum, as well as alloys of various metals,and other conducting materials, such as, carbon, including glassycarbon, reticulated vitreous carbon, basal plane graphite, edge planegraphite and graphite. Doped oxides such as indium-tin oxide andsemiconductors such as silicon oxide and gallium arsenide are alsocontemplated. Additionally, the electrodes may be composed of conductingpolymers, metal doped polymers, conducting ceramics and conductingclays. Among the metals, platinum and palladium are especially preferredbecause of the advantageous properties associated with their ability toabsorb hydrogen, i.e., their ability to be “preloaded” with hydrogenbefore being used in the methods of the invention.

The electrodes may be connected to an electric source in any knownmanner. Preferred ways of connecting the electrodes to the electricsource include CMOS (complementary metal oxide semiconductor) switchingcircuitry, radio and microwave frequency addressable switches, lightaddressable switches, direct connection from an electrode to a bond padon the perimeter of a semiconductor chip, and combinations thereof. CMOSswitching circuitry involves the connection of each of the electrodes toa CMOS transistor switch. The switch could be accessed by sending anelectronic address signal down a common bus to SRAM (static randomaccess memory) circuitry associated with each electrode. When the switchis “on”, the electrode is connected to an electric source. Radio andmicrowave frequency addressable switches involve the electrodes beingswitched by a RF or microwave signal. This allows the switches to bethrown both with and/or without using switching logic. The switches canbe tuned to receive a particular frequency or modulation frequency andswitch without switching logic. Light addressable switches are switchedby light. In this method, the electrodes can also be switched with andwithout switching logic. The light signal can be spatially localized toafford switching without switching logic. This could be accomplished,for example, by scanning a laser beam over the electrode array; theelectrode being switched each time the laser illuminates it.

In some aspects, a predefined region can be achieved by physicallyseparating the regions (i.e., beads, resins, gels, etc.) into wells,trays, etc.

A “protecting group” is a moiety which is bound to a molecule anddesigned to block one reactive site in a molecule, but may be spatiallyremoved upon selective exposure to an activator or a deprotectingreagent. Several examples of protecting groups are known in theliterature. The proper selection of protecting group (also known asprotective group) for a particular synthesis would be governed by theoverall methods employed in the synthesis. Activators include, forexample, electromagnetic radiation, ion beams, electric fields, magneticfields, electron beams, x-ray, and the like. A deprotecting reagentcould include, for example, an acid, a base or a free radical.Protective groups are materials that bind to a monomer, a linkermolecule or a pre-formed molecule to protect a reactive functionality onthe monomer, linker molecule or pre-formed molecule, which may beremoved upon selective exposure to an activator, such as anelectrochemically generated reagent. Protective groups that may be usedin accordance with an embodiment of the invention preferably include allacid and base labile protecting groups. For example, peptide aminegroups are preferably protected by t-butyloxycarbonyl (BOC) orbenzyloxycarbonyl (CBZ), both of which are acid labile, or by9-fluorenylmethoxycarbonyl (FMOC), which is base labile. Additionally,hydroxyl groups on phosphoramidites may be protected by dimethoxytrityl(DMT), which is acid labile. Exocyclic amine groups on nucleosides, inparticular on phosphoramidites, are preferably protected bydimethylformamidine on the adenosine and guanosine bases, and isobutyrylon the cytidine bases, both of which are base labile protecting groups.This protection strategy is known as fast oligonucleotide deprotection(FOD).

Any unreacted deprotected chemical functional groups may be capped atany point during a synthesis reaction to avoid or to prevent furtherbonding at such molecule. Capping groups “cap” deprotected functionalgroups by, for example, binding with the unreacted amino functions toform amides. Capping agents suitable for use in an embodiment of theinvention include: acetic anhydride, n-acetylimidizole, isopropenylformate, fluorescamine, 3-nitrophthalic anhydride and 3-sulfoproponicanhydride. Of these, acetic anhydride and n-acetylimidizole arepreferred.

Additional protecting groups that may be used in accordance with anembodiment of the invention include acid labile groups for protectingamino moieties: tertbutyloxycarbonyl,-tert-amyloxycarbonyl,adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl,2-(p-biphenyl)propyl(2)oxycarbonyl,2-(p-phenylazophenylyl)propyl(2)oxycarbonyl,alpha.,.alpha.-dimethyl-3,5-dimethyloxybenzyloxy-carbonyl,2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl,benzyloxycarbonyl, furfuryloxycarbonyl, triphenylmethyl (trityl),p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl,diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl,and 1-naphthylidene; as base labile groups for protecting aminomoieties: 9-fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl,and 5-benzisoazolylmethyleneoxycarbonyl; as groups for protecting aminomoieties that are labile when reduced: dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; as groups for protecting aminomoieties that are labile when oxidized: (ethylthio)carbonyl; as groupsfor protecting amino moieties that are labile to miscellaneous reagents,the appropriate agent is listed in parenthesis after the group:phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl(2-aminothiophenol); acid labile groups for protecting carboxylic acids:tert-butyl ester; acid labile groups for protecting hydroxyl groups:dimethyltrityl; and basic labile groups for protecting phosphotriestergroups: cyanoethyl.

An “electrochemical reagent” refers to a chemical generated at aselected electrode by applying a sufficient electrical potential to theselected electrode and is capable of electrochemically removing aprotecting group from a chemical functional group. The chemical groupwould generally be attached to a molecule. Removal of a protectinggroup, or “deprotection,” in accordance with the invention, preferablyoccurs at a particular portion of a molecule when a chemical reagentgenerated by the electrode acts to deprotect or remove, for example, anacid or base labile protecting group from the molecule. Thiselectrochemical deprotection reaction may be direct, or may involve oneor more intermediate chemical reactions that are ultimately driven orcontrolled by the imposition of sufficient electrical potential at aselected electrode.

Electrochemical reagents that can be generated electrochemically at anelectrode fall into two broad classes: oxidants and reductants. Oxidantsthat can be generated electrochemically, for example, include iodine,iodate, periodic acid, hydrogen peroxide, hypochlorite, metavanadate,bromate, dichromate, cerium (IV), and permanganate ions. Reductants thatcan be generated electrochemically, for example, include chromium (II),ferrocyanide, thiols, thiosulfate, titanium (III), arsenic (III) andiron (II) ions. The miscellaneous reagents include bromine, chloride,protons and hydroxyl ions. Among the foregoing reagents, protons,hydroxyl, iodine, bromine, chlorine and thiol ions are preferred.

The generation of and electrochemical reagent of a type of chemicalspecies requires that the electric potential of the electrode thatgenerates the electrochemical reagent have a certain value, which may beachieved by specifying either the voltage or the current. There are twoways to achieve the potential at this electrode: either the voltage maybe specified at a value or the current can be determined such that it issufficient to provide a voltage. The range between the minimum andmaximum potential values could be determined by the type ofelectrochemical reagent chosen to be generated.

An “activating group” refers to those groups which, when attached to aparticular chemical functional group or reactive site, render that sitemore reactive toward covalent bond formation with a second chemicalfunctional group or reactive site.

A “polymeric brush” ordinarily refers to polymer films comprising chainsof polymers that are attached to the surface of a substrate. Thepolymeric brush could be a functionalized polymer films which comprisefunctional groups such as hydroxyl, amino, carboxyl, thiol, amide,cyanate, thiocyanate, isocyanate and isothio cyanate groups, or acombination thereof, on the polymer chains at one or more predefinedregions. The polymeric brushes of the embodiment of the invention arecapable of attachment or stepwise synthesis of macromolecules thereon.

A “linker” molecule refers to any of those molecules described supra andpreferably should be about 4 to about 40 atoms long to providesufficient exposure. The linker molecules may be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units,diamines, diacids, amino acids, among others, and combinations thereof.Alternatively, the linkers may be the same molecule type as that beingsynthesized (i.e., nascent polymers), such as polynucleotides,oligopeptides, or oligosaccharides.

The linker molecule or substrate itself and monomers used herein areprovided with a functional group to which is bound a protective group.Generally, the protective group is on the distal or terminal end of amolecule. Preferably, the protective group is on the distal or terminalend of the linker molecule opposite the substrate. The protective groupmay be either a negative protective group (i.e., the protective grouprenders the linker molecules less reactive with a monomer upon exposure)or a positive protective group (i.e., the protective group renders thelinker molecules more reactive with a monomer upon exposure). In thecase of negative protective groups, there could be an additional step ofreactivation. In some embodiments, this will be done by heating.

The polymeric brush or the linker molecule may be provided with acleavable group at an intermediate position, which group can be cleavedwith an electrochemically generated reagent. This group is preferablycleaved with a reagent different from the reagent(s) used to remove theprotective groups. This enables removal of the various synthesizedpolymers or nucleic acid sequences following completion of thesynthesis. The cleavable group could be acetic anhydride,n-acetylimidizole, isopropenyl formate, fluorescamine, 3-nitrophthalicanhydride and 3-sulfoproponic anhydride. Of these, acetic anhydride andn-acetylimidizole are preferred.

The polymeric brush or the linker molecule could be of sufficient lengthto permit polymers on a completed substrate to interact freely withbinding entities (monomers, for example) exposed to the substrate. Thepolymeric brush or the linker molecule, when used, could preferably belong enough to provide sufficient exposure of the functional groups tothe binding entity. The linker molecules may include, for example, arylacetylene, ethylene glycol oligomers containing from 2 to 20 monomerunits, diamines, diacids, amino acids, and combinations thereof. Otherlinker molecules may be used in accordance with the differentembodiments of the present invention and will be recognized by thoseskilled in the art in light of this disclosure. In one embodiment,derivatives of the acid labile 4,4′-dimethyoxytrityl molecules with anexocyclic active ester can be used in accordance with an embodiment ofthe invention. More preferably,N-succinimidyl-4[bis-(4-methoxyphenyl)-chloromethyl]-benzoate is used asa cleavable linker molecule during DNA synthesis. Alternatively, othermanners of cleaving can be used over the entire array at the same time,such as chemical reagents, light or heat.

“Monomer” as used herein refers to those monomers that are used to aform a polymer. However, the meaning of the monomer will be clear fromthe context in which it is used. The monomers in a given polymer ormacromolecule can be identical to or different from each other. Amonomer can be a small or a large molecule, regardless of molecularweight. Furthermore, each of the monomers may be protected members whichare modified after synthesis.

The monomers for forming the polymers of the embodiments of theinvention, e.g., a polymeric brush or a linker molecule, have forexample the general structure:

wherein R¹ is hydrogen or lower alkyl; R₂ and R₃ are independentlyhydrogen, or -Y-Z, wherein Y is lower alkyl, and Z is hydroxyl, amino,or C(O)—R, where R is hydrogen, lower alkoxy or aryloxy.

The term “alkyl” refers to those groups such as methyl, ethyl, propyl,butyl etc, which may be linear, branched or cyclic.

The term “alkoxy” refers to groups such as methoxy, ethoxy, propoxy,butoxy, etc., which may be linear, branched or cyclic.

The term “lower” as used in the context of lower alkyl or lower alkoxyrefers to groups having one to six carbons.

The term “aryl” refers to an aromatic hydrocarbon ring to which isattached an alkyl group. The term “aryloxy” refers to an aromatichydrocarbon ring to which is attached an alkoxy group. One of ordinaryskill in the art would readily understand these terms.

Other monomers for preparing macromolecules of the embodiments of theinvention are well-known in the art. For example, when the macromoleculeis a peptide, the monomers include, but are not restricted to, forexample, amino acids such as the L-amino acids, the D-amino acids, andthe synthetic and/or natural amino acids. When the macromolecule is anucleic acid, or polynucleotide, the monomers include any nucleotide.When the macromolecule is a polysaccharide, the monomers can be anypentose, hexose, heptose, or their derivatives.

A “macromolecule” or “polymer” comprises two or more monomers covalentlyjoined. The monomers may be joined one at a time or in strings ofmultiple monomers, ordinarily known as “oligomers.” Thus, for example,one monomer and a string of five monomers may be joined to form amacromolecule or polymer of six monomers. Similarly, a string of fiftymonomers may be joined with a string of hundred monomers to form amacromolecule or polymer of one hundred and fifty monomers. The termpolymer as used herein includes, for example, both linear and cyclicpolymers of nucleic acids, polynucleotides, polynucleotides,polysaccharides, oligosaccharides, proteins, polypeptides, peptides,phospholipids and peptide nucleic acids (PNAs). The peptides includethose peptides having either α-, β-, or ω-amino acids. In addition,polymers include heteropolymers in which a known drug is covalentlybound to any of the above, polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, polyacetates, or other polymers which will beapparent upon review of this disclosure.

A “nanomaterial” as used herein refers to a structure, a device or asystem having a dimension at the atomic, molecular or macromolecularlevels, in the length scale of approximately 1-500 nanometer range.Preferably, a nanomaterial has properties and functions because of thesize and can be manipulated and controlled on the atomic level.

A “carbon nanotube” refers to a fullerene molecule having a cylindricalor toroidal shape. A “fullerene” refers to a form of carbon having alarge molecule consisting of an empty cage of sixty or more carbonatoms.

The term “nucleotide” includes deoxynucleotides and analogs thereof.These analogs are those molecules having some structural features incommon with a naturally occurring nucleotide such that when incorporatedinto a polynucleotide sequence, they allow hybridization with acomplementary polynucleotide in solution. Typically, these analogs arederived from naturally occurring nucleotides by replacing and/ormodifying the base, the ribose or the phosphodiester moiety. The changescan be tailor-made to stabilize or destabilize hybrid formation, or toenhance the specificity of hybridization with a complementarypolynucleotide sequence as desired, or to enhance stability of thepolynucleotide.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides, that comprise purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. Polynucleotides of the embodiments of theinvention include sequences of deoxyribopolynucleotide (DNA),ribopolynucleotide (RNA), or DNA copies of ribopolynucleotide (cDNA)which may be isolated from natural sources, recombinantly produced, orartificially synthesized. A further example of a polynucleotide of theembodiments of the invention may be polyamide polynucleotide (PNA). Thepolynucleotides and nucleic acids may exist as single-stranded ordouble-stranded. The backbone of the polynucleotide can comprise sugarsand phosphate groups, as may typically be found in RNA or DNA, ormodified or substituted sugar or phosphate groups. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. The polymers made of nucleotides such asnucleic acids, polynucleotides and polynucleotides may also be referredto herein as “nucleotide polymers.

An “oligonucleotide” is a polynucleotide having 2 to 20 nucleotides.Phosphoramidites protected in this manner are known as FODphosphoramidites.

Analogs also include protected and/or modified monomers as areconventionally used in polynucleotide synthesis. As one of skill in theart is well aware, polynucleotide synthesis uses a variety ofbase-protected nucleoside derivatives in which one or more of thenitrogens of the purine and pyrimidine moiety are protected by groupssuch as dimethoxytrityl, benzyl, tert-butyl, isobutyl and the like.

For instance, structural groups are optionally added to the ribose orbase of a nucleoside for incorporation into a polynucleotide, such as amethyl, propyl or allyl group at the 2′-O position on the ribose, or afluoro group which substitutes for the 2′-O group, or a bromo group onthe ribonucleoside base. 2′-O-methyloligoribonucleotides (2′-O-MeORNs)have a higher affinity for complementary polynucleotides (especiallyRNA) than their unmodified counterparts. Alternatively, deazapurines anddeazapyrimidines in which one or more N atoms of the purine orpyrimidine heterocyclic ring are replaced by C atoms can also be used.

The phosphodiester linkage or “sugar-phosphate backbone” of thepolynucleotide can also be substituted or modified, for instance withmethyl phosphonates, O-methyl phosphates or phosphororthioates. Anotherexample of a polynucleotide comprising such modified linkages forpurposes of this disclosure includes “peptide polynucleotides” in whicha polyamide backbone is attached to polynucleotide bases, or modifiedpolynucleotide bases. Peptide polynucleotides which comprise a polyamidebackbone and the bases found in naturally occurring nucleotides arecommercially available.

Nucleotides with modified bases can also be used in the embodiments ofthe invention. Some examples of base modifications include2-aminoadenine, 5-methylcytosine, 5-(propyn-1-yl)cytosine,5-(propyn-1-yl)uracil, 5-bromouracil, 5-bromocytosine,hydroxymethylcytosine, methyluracil, hydroxymethyluracil, anddihydroxypentyluracil which can be incorporated into polynucleotides inorder to modify binding affinity for complementary polynucleotides.

Groups can also be linked to various positions on the nucleoside sugarring or on the purine or pyrimidine rings which may stabilize the duplexby electrostatic interactions with the negatively charged phosphatebackbone, or through interactions in the major and minor groves. Forexample, adenosine and guanosine nucleotides can be substituted at theN² position with an imidazolyl propyl group, increasing duplexstability. Universal base analogues such as 3-nitropyrrole and5-nitroindole can also be included. A variety of modifiedpolynucleotides suitable for use in the embodiments of the invention aredescribed in the literature.

When the macromolecule of interest is a peptide, the amino acids can beany amino acids, including α, β, or ω-amino acids. When the amino acidsare α-amino acids, either the L-optical isomer or the D-optical isomermay be used. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also contemplated by theembodiments of the invention. These amino acids are well-known in theart.

An “antibody” is any of various bodies or substances in the blood whichact in antagonism to harmful foreign bodies, as toxins or the bacteriaproducing the toxins. Normal blood serum apparently contains variousantibodies, and the introduction of toxins or of foreign cells alsoresults in the development of their specific antibodies. For example, anantibody is a Y-shaped protein on the surface of B cells that issecreted into the blood or lymph in response to an antigenic stimulus,such as a bacterium, virus, parasite, or transplanted organ, and thatneutralizes the antigen by binding specifically to it; animmunoglobulin.

A “peptide” is a polymer in which the monomers are amino acids and whichare joined together through amide bonds and alternatively referred to asa polypeptide. In the context of this specification it should beappreciated that the amino acids may be the L-optical isomer or theD-optical isomer. Peptides are two or more amino acid monomers long, andoften more than 20 amino acid monomers long.

A “protein” is a long polymer of amino acids linked via peptide bondsand which may be composed of two or more polypeptide chains. Morespecifically, the term “protein” refers to a molecule composed of one ormore chains of amino acids in a specific order; for example, the orderas determined by the base sequence of nucleotides in the gene coding forthe protein. Proteins are essential for the structure, function, andregulation of the body's cells, tissues, and organs, and each proteinhas unique functions. Examples are hormones, enzymes, and antibodies.

A “carbohydrate” is a compound with carbon, hydrogen and oxygen usuallyin a proportion to form water with the general formula C_(n)(H₂O)_(n).Carbohydrates can also be called chemically as neutral compounds ofcarbon, hydrogen and oxygen. Carbohydrates are mainly sugars andstarches, together constituting one of the three principal types ofnutrients used as energy sources (calories) by the body. Carbohydratescome in simple forms such as sugars and in complex forms such asstarches and fiber. The body breaks down most sugars and starches intoglucose, a simple sugar that the body can use to feed its cells. Complexcarbohydrates are derived from plants. Dietary intake of complexcarbohydrates can lower blood cholesterol when they are substituted forsaturated fat. Carbohydrates are classified into mono, di, tri, poly andheterosaccharides. The smallest carbohydrates are monosaccharides suchas glucose whereas polysaccharides such as starch, cellulose andglycogen can be large and even indeterminate in length.

A “lipid” is defined as a substance such as a fat, oil or wax thatdissolves in alcohol but not in water. Lipids contain carbon, hydrogenand oxygen but have far less oxygen proportionally than carbohydrates.Lipids are an important part of living cells. Together withcarbohydrates and proteins, lipids are the main constituents of plantand animal cells. Cholesterol and triglycerides are lipids. Lipids areeasily stored in the body. They serve as a source of fuel and are animportant constituent of the structure of cells. Lipids include fattyacids, neutral fats, waxes and steroids (like cortisone). Compoundlipids (lipids complexed with another type of chemical compound)comprise the lipoproteins, glycolipids and phospholipids.

An “antigen” a substance that is capable of causing the production of anantibody. For example, when an antigen is introduced into the body, itstimulates the production of an antibody. Antigens include toxins,bacteria, foreign blood cells, and the cells of transplanted organs.

The term “sequence” refers to the particular ordering of monomers withina macromolecule and it may be referred to herein as the sequence of themacromolecule.

The term “hybridization” refers to the process in which twosingle-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” For example, hybridization refers to theformation of hybrids between a probe polynucleotide (e.g., apolynucleotide of the invention which may include substitutions,deletion, and/or additions) and a specific target polynucleotide (e.g.,an analyte polynucleotide) wherein the probe preferentially hybridizesto the specific target polynucleotide and substantially does nothybridize to polynucleotides consisting of sequences which are notsubstantially complementary to the target polynucleotide. However, itwill be recognized by those of skill that the minimum length of apolynucleotide desired for specific hybridization to a targetpolynucleotide will depend on several factors: G/C content, positioningof mismatched bases (if any), degree of uniqueness of the sequence ascompared to the population of target polynucleotides, and chemicalnature of the polynucleotide (e.g., methylphosphonate backbone,phosphorothiolate, etc.), among others.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known in the art.

It is appreciated that the ability of two single strandedpolynucleotides to hybridize will depend upon factors such as theirdegree of complementarity as well as the stringency of the hybridizationreaction conditions.

As used herein, “stringency” refers to the conditions of a hybridizationreaction that influence the degree to which polynucleotides hybridize.Stringent conditions can be selected that allow polynucleotide duplexesto be distinguished based on their degree of mismatch. High stringencyis correlated with a lower probability for the formation of a duplexcontaining mismatched bases. Thus, the higher the stringency, thegreater the probability that two single-stranded polynucleotides,capable of forming a mismatched duplex, will remain single-stranded.Conversely, at lower stringency, the probability of formation of amismatched duplex is increased.

The appropriate stringency that will allow selection of aperfectly-matched duplex, compared to a duplex containing one or moremismatches (or that will allow selection of a particular mismatchedduplex compared to a duplex with a higher degree of mismatch) isgenerally determined empirically. Means for adjusting the stringency ofa hybridization reaction are well-known to those of skill in the art.

A “ligand” is a molecule that is recognized by a particular receptor.Examples of ligands that can be investigated by this invention include,but are not restricted to, agonists and antagonists for cell membranereceptors, toxins and venoms, viral epitopes, hormones, hormonereceptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g.opiates, steroids, etc.), lectins, sugars, polynucleotides, nucleicacids, oligosaccharides, proteins, and monoclonal antibodies. Ligands tocells or cell-derived molecules, which can include both known andunknown ligands as well as putative drug candidates that are eitherunattached to other solid supports or attached to surfaces orparticle-like structures, could interact with other cell-derivedmolecules in a manner such that binding between two binding partnersoccurs and can be detected. One of the binding partners or its attachedsupport can additionally be derivatized with a substance that can berecognized and quantified by a detection apparatus. This complex(through interaction) is then brought into the presence of the detectionapparatus using characteristics of the associated complex thatdifferentiate it from the unassociated binding partners.

An “affinity binding partner” or “binding partner” could be a probe or aligand defined above.

A “receptor” is molecule that has an affinity for a given ligand.Receptors may-be naturally-occurring or manmade molecules. Also, theycan be employed in their unaltered state or as aggregates with otherspecies. Receptors may be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of receptors which can be employed by this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. However, as theterm receptor is used herein, no difference in meaning is intended. A“ligand receptor pair” is formed when two macromolecules have combinedthrough molecular recognition to form a complex. Other examples ofreceptors which can be investigated by this invention include but arenot restricted to:

a) Microorganism receptors: Determination of ligands which bind toreceptors, such as specific transport proteins or enzymes essential tosurvival of microorganisms, is useful in developing a new class ofantibiotics. Of particular value would be antibiotics againstopportunistic fungi, protozoa, and those bacteria resistant to theantibiotics in current use.

b) Enzymes: For instance, one type of receptor is the binding site ofenzymes such as the enzymes responsible for cleaving neurotransmitters;determination of ligands which bind to certain receptors to modulate theaction of the enzymes which cleave the different neurotransmitters isuseful in the development of drugs which can be used in the treatment ofdisorders of neurotransmission.

c) Antibodies: For instance, the invention may be useful ininvestigating the ligand-binding site on the antibody molecule whichcombines with the epitope of an antigen of interest; determining asequence that mimics an antigenic epitope may lead to the-development ofvaccines of which the immunogen is based on one or more of suchsequences or lead to the development of related diagnostic agents orcompounds useful in therapeutic treatments such as for auto-immunediseases (e.g., by blocking the binding of the “anti-self” antibodies).

d) Nucleic Acids: Sequences of nucleic acids may be synthesized toestablish DNA or RNA binding sequences.

e) Catalytic Polypeptides: Polymers, preferably polypeptides, which arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products. Such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,which functionality is capable of chemically modifying the boundreactant.

f) Hormone receptors: Examples of hormones receptors include, e.g., thereceptors for insulin and growth hormone. Determination of the ligandswhich bind with high affinity to a receptor is useful in the developmentof, for example, an oral replacement of the daily injections whichdiabetics take to relieve the symptoms of diabetes. Other examples arethe vasoconstrictive hormone receptors; determination of those ligandswhich bind to a receptor may lead to the development of drugs to controlblood pressure.

g) Opiate receptors: Determination of ligands which bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

By “analyte” is meant any molecule or compound. An analyte can be in thesolid, liquid, gaseous or vapor phase. By “gaseous or vapor phaseanalyte” is meant a molecule or compound that is present, for example,in the headspace of a liquid, in ambient air, in a breath sample, in agas, or as a contaminant in any of the foregoing. It will be recognizedthat the physical state of the gas or vapor phase can be changed bypressure, temperature as well as by affecting surface tension of aliquid by the presence of or addition of salts etc.

The term analyte further includes polynucleotide analytes such as thosepolynucleotides defined below. These include m-RNA, r-RNA, t-RNA, DNA,DNA-RNA duplexes, etc. The term analyte also includes receptors that arepolynucleotide binding agents, such as, for example, peptide nucleicacids (PNA), restriction enzymes, activators, repressors, nucleases,polymerases, histones, repair enzymes, chemotherapeutic agents, and thelike.

The analyte may be a molecule found directly in a sample such as a bodyfluid from a host. The sample can be examined directly or may bepretreated to render the analyte more readily detectible. Furthermore,the analyte of interest may be determined by detecting an agentprobative of the analyte of interest such as a specific binding pairmember complementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay. The body fluid can be, for example, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, and the like.

The analyte can further be a member of a specific binding pair (sbp) andmay be a ligand, which is monovalent (monoepitopic) or polyvalent(polyepitopic), usually antigenic or haptenic, and is a single compoundor plurality of compounds which share at least one common epitopic ordeterminant site. The analyte can be a part of a cell such as bacteriaor a cell bearing a blood group antigen such as A, B, D, etc., or an HLAantigen or a microorganism, e.g., bacterium, fungus, protozoan, orvirus. Also, the analyte could be charged. A member of a specificbinding pair (“sbp member”) is one of two different molecules, having anarea on the surface or in a cavity which specifically binds to and isthereby defined as complementary with a particular spatial and polarorganization of the other molecule. The members of the specific bindingpair are referred to as ligand and receptor (antiligand) or analyte andprobe. Therefore, a probe is a molecule that specifically binds ananalyte. These will usually be members of an immunological pair such asantigen-antibody, although other specific binding pairs such asbiotin-avidin, hormones-hormone receptors, nucleic acid duplexes,IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA, and thelike are not immunological pairs but are included in the invention andthe definition of sbp member.

Bioanalyte can also be complex of molecules or compounds in organized orrandom fashion, such cells, virus, bacteria, fungi, etc.

“Specific binding” is the specific recognition of one of two differentmolecules for the other compared to substantially less recognition ofother molecules. Generally, the molecules have areas on their surfacesor in cavities giving rise to specific recognition between the twomolecules. Exemplary of specific binding are antibody-antigeninteractions, enzyme—substrate interactions, polynucleotidehybridization interactions, and so forth.

“Non-specific binding” is non-covalent binding between molecules that isrelatively independent of specific surface structures. Non-specificbinding may result from several factors including hydrophobicinteractions between molecules.

The polyvalent ligand analytes will normally be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations include components of bacteria,viruses, chromosomes, genes, mitochondria, nuclei, cell membranes andthe like.

For the most part, the polyepitopic ligand analytes can have a molecularweight of at least about 5,000, more usually at least about 10,000. Inthe poly(amino acid) category, the poly(amino acids) of interest willgenerally be from about 5,000 to 5,000,000 molecular weight, moreusually from about 20,000 to 1,000,000 molecular weight; among thehormones of interest, the molecular weights will usually range fromabout 5,000 to 60,000 molecular weight.

The monoepitopic ligand analytes can generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.The analytes include drugs, metabolites, pesticides, pollutants, and thelike. Included among drugs of interest are the alkaloids. Among thealkaloids are morphine alkaloids, which includes morphine, codeine,heroin, dextromethorphan, their derivatives and metabolites; cocainealkaloids, which include cocaine and benzyl ecgonine, their derivativesand metabolites; ergot alkaloids, which include the diethylamide oflysergic acid; steroid alkaloids; iminazoyl alkaloids; quinazolinealkaloids; isoquinoline alkaloids; quinoline alkaloids, which includequinine and quinidine; diterpene alkaloids, their derivatives andmetabolites.

The term “reporter” means a detectable moiety. The reporter can bedetected, for example, by Raman spectroscopy. Generally, the reporterand any molecule linked to the reporter can be detected without a secondbinding reaction. The reporter can be COIN (composite-organic-inorganicnanoparticle), magnetic-COIN, quantum dots, and other Raman orfluorescent tags, for example.

The term “COIN” refers to a composite-organic-inorganic nanoparticle(s).The COIN could be surface-enhanced Raman spectroscopy (SERS)-activenanoparticles incorporated into a gel matrix and used in certain otheranalyte separation techniques described herein. COINs are compositeorganic-inorganic nanoparticles. These SERS-active probe constructscomprise a core and a surface, wherein the core comprises a metalliccolloid comprising a first metal and a Raman-active organic compound.The COINs can further comprise a second metal different from the firstmetal, wherein the second metal forms a layer overlying the surface ofthe nanoparticle. The COINs can further comprise an organic layeroverlying the metal layer, which organic layer comprises the probe.Suitable probes for attachment to the surface of the SERS-activenanoparticles include, without limitation, antibodies, antigens,polynucleotides, oligonucleotides, receptors, ligands, and the like.

The metal required for achieving a suitable SERS signal is inherent inthe COIN, and a wide variety of Raman-active organic compounds can beincorporated into the particle. Indeed, a large number of unique Ramansignatures can be created by employing nanoparticles containingRaman-active organic compounds of different structures, mixtures, andratios. Thus, the methods described herein employing COINs are usefulfor the simultaneous detection of many analytes in a sample, resultingin rapid qualitative analysis of the contents of “profile” of a bodyfluid. In addition, since many COINs can be incorporated into a singlenanoparticle, the SERS signal from a single COIN particle is strongrelative to SERS signals obtained from Raman-active materials that donot contain the nanoparticles described herein as COINs. This situationresults in increased sensitivity compared to Raman-techniques that donot utilize COINs.

COINs could be prepared using standard metal colloid chemistry. Thepreparation of COINs also takes advantage of the ability of metals toadsorb organic compounds. Indeed, since Raman-active organic compoundsare adsorbed onto the metal during formation of the metallic colloids,many Raman-active organic compounds can be incorporated into the COINwithout requiring special attachment chemistry.

In general, the COINs could be prepared as follows. An aqueous solutionis prepared containing suitable metal cations, a reducing agent, and atleast one suitable Raman-active organic compound. The components of thesolution are then subject to conditions that reduce the metallic cationsto form neutral, colloidal metal particles. Since the formation of themetallic colloids occurs in the presence of a suitable Raman-activeorganic compound, the Raman-active organic compound is readily adsorbedonto the metal during colloid formation. This COIN can typically beisolated by membrane filtration. In addition, COINs of different sizescan be enriched by centrifugation.

The COINs can include a second metal different from the first metal,wherein the second metal forms a layer overlying the surface of thenanoparticle. To prepare SERS-active nanoparticle, COINs are placed inan aqueous solution containing suitable second metal cations and areducing agent. The components of the solution are then subject toconditions that reduce the second metallic cations so as to form ametallic layer overlying the surface of the nanoparticle. In certainembodiments, the second metal layer includes metals, such as, forexample, silver, gold, platinum, aluminum, and the like. These COINs canbe isolated and or enriched in the same manner as explained below.Typically, COINs are substantially spherical and range in size fromabout 20 nm to 60 nm. The size of the nanoparticle is selected to beabout one-half the wavelength of light used to irradiate the COINsduring detection.

Typically, organic compounds of COINs are attached to a layer of asecond metal by covalently attaching organic compounds to the surface ofthe metal layer Covalent attachment of an organic layer to the metalliclayer can be achieved in a variety ways well known to those skilled inthe art, such as for example, through thiol-metal bonds. In alternativeembodiments, the organic molecules attached to the metal layer can becrosslinked to form a molecular network.

The COIN(s) can include cores containing magnetic materials, such as,for example, iron oxides, and the like such that the COIN is a magneticCOIN. Magnetic COINs can be handled without centrifugation usingcommonly available magnetic particle handling systems. Indeed, magnetismcan be used as a mechanism for separating biological targets attached tomagnetic COIN particles tagged with particular biological probes.

As used herein, “Raman-active organic compound” refers to an organicmolecule that produces a unique SERS signature in response to excitationby a laser. A variety of Raman-active organic compounds are contemplatedfor use as components in COINs. In certain embodiments, Raman-activeorganic compounds are polycyclic aromatic or heteroaromatic compounds.Typically the Raman-active organic compound has a molecular weight lessthan about 300 Daltons.

Additional, non-limiting examples of Raman-active organic compoundsuseful in COINs include TRIT (tetramethyl rhodamine isothiol), NBD(7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blueviolet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine,biotin, digoxigenin, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxy rhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino phthalocyanines, azomethines, cyanines,xanthines, succinylfluoresceins, aminoacridine, and the like.

In certain embodiments, the Raman-active compound is adenine, adenine,4-amino-pyrazolo(3,4-d)pyrimidine, 2-fluoroadenine, N6-benzolyadenine,kinetin, dimethyl-allyl-amino-adenine, zeatin, bromo-adenine,8-aza-adenine, 8-azaguanine, 6-mercaptopurine,4-amino-6-mercaptopyrazolo(3,4-d)pyrimidine, 8-mercaptoadenine, or9-amino-acridine 4-amino-pyrazolo(3,4-d)pyrimidine, or 2-fluoroadenine.In one embodiment, the Raman-active compound is adenine.

When “fluorescent compounds” are incorporated into COINs, thefluorescent compounds can include, but are not limited to, dyes,intrinsically fluorescent proteins, lanthanide phosphors, and the like.Dyes useful for incorporation into COINs include, for example, rhodamineand derivatives, such as Texas Red, ROX (6-carboxy-X-rhodamine),rhodamine-NHS, and TAMRA (5/6-carboxytetramethyl rhodamine NHS);fluorescein and derivatives, such as 5-bromomethyl fluorescein and FAM(5′-carboxyfluorescein NHS), Lucifer Yellow, IAEDANS, 7-Me₂,N-coumarin-4-acetate, 7-OH-4-CH₃-coumarin-3-acetate,7-NH₂-4CH₃-coumarin-3-acetate (AMCA), monobromobimane, pyrenetrisulfonates, such as Cascade Blue, andmonobromotrimethyl-ammoniobimane.

Multiplex testing of a complex sample would generally be based on acoding system that possesses identifiers for a large number of reactantsin the sample. The primary variable that determines the achievablenumbers of identifiers in currently known coding systems is, however,the physical dimension. Tagging techniques, based on surface-enhancedRaman scattering (SERS) of fluorescent dyes, could be used in theembodiments of this invention for developing chemical structure-basedcoding systems. The organic compound-assisted metal fusion (OCAM) methodcould be used to produce composite organic-inorganic nanoparticles(COIN) that are highly effective in generating SERS signals allowssynthesis of COIN labels from a wide range of organic compounds toproduce sufficient distinguishable COIN Raman signatures to assay anycomplex biological sample. Thus COIN particles may be used as a codingsystem for multiplex and amplification-free detection of bioanalytes atnear single molecule levels.

COIN particles generate intrinsic SERS signal without additionalreagents. Using the OCAMF-based COIN synthesis chemistry, it is possibleto generate a large number of different COIN signatures by mixing alimited number of Raman labels for use in multiplex assays in differentratios and combinations. In a simplified scenario, the Raman spectrum ofa sample labeled with COIN particles may be characterized by threeparameters: (a) peak position (designated as L), which depends on thechemical structure of Raman labels used and the umber of availablelabels, (b) peak number (designated as Al), which depends on the numberof labels used together in a single COIN, and (c) peak height(designated as i), which depends on the ranges of relative peakintensity.

The total number of possible distinguishable Raman signatures(designated as 7) may be calculated from the following equation:

$T = {\sum\limits_{k = 1}^{M}{\frac{L!}{{\left( {L - k} \right)!}{k!}}{P\left( {i,k} \right)}}}$

where P(i, k)=i^(k)−i+1, being the intensity multiplier which representsthe number of distinct Raman spectra that may be generated by combiningk (k=1 to M) labels for a given i value. The multiple organic compoundsmay be mixed in various combinations, numbers and ratios to make themultiple distinguishable Raman signatures. It has been shown thatspectral signatures having closely positioned peaks (15 cm⁻¹) may beresolved visually. Theoretically, over a million of Raman signatures maybe made within the Raman shift range of 500-2000 cm⁻¹ by incorporatingmultiple organic molecules into COIN as Raman labels using theOCAMF-based COIN synthesis chemistry.

Thus, OCAMF chemistry allows incorporation of a wide range of Ramanlabels into metal colloids to perform parallel synthesis of a largenumber of COIN labels with distinguishable Raman signatures in a matterof hours by mixing several organic Raman-active compounds of differentstructures, mixtures, and ratios for use in the invention methodsdescribed herein.

COINs may be used to detect the presence of a particular target analyte,for example, a nucleic acid, oligonucleotide, protein, enzyme, antibodyor antigen. The nanoparticles may also be used to screen bioactiveagents, i.e. drug candidates, for binding to a particular target or todetect agents like pollutants. Any analyte for which a probe moiety,such as a peptide, protein, oligonucleotide or aptamer, may be designedcan be used in combination with the disclosed nanoparticles.

Also, SERS-active COINs that have an antibody as binding partner couldbe used to detect interaction of the Raman-active antibody labeledconstructs with antigens either in solution or on a solid support. Itwill be understood that such immunoassays can be performed using knownmethods such as are used, for example, in ELISA assays, Westernblotting, or protein arrays, utilizing a SERS-active COIN having anantibody as the probe and acting as either a primary or a secondaryantibody, in place of a primary or secondary antibody labeled with anenzyme or a radioactive compound. In another example, a SERS-active COINis attached to an enzyme probe for use in detecting interaction of theenzyme with a substrate.

Another group of exemplary methods could use the SERS-active COINs todetect a target nucleic acid. Such a method is useful, for example, fordetection of infectious agents within a clinical sample, detection of anamplification product derived from genomic DNA or RNA or message RNA, ordetection of a gene (cDNA) insert within a clone. For certain methodsaimed at detection of a target polynucleotide, an oligonucleotide probeis synthesized using methods known in the art. The oligonucleotide isthen used to functionalize a SERS-active COIN. Detection of the specificRaman label in the SERS-active COIN identifies the nucleotide sequenceof the oligonucleotide probe, which in turn provides informationregarding the nucleotide sequence of the target polynucleotide.

A “quantum dot” is a particle of matter so small that the addition orremoval of an electron changes its properties in some useful way. Allatoms are, of course, quantum dots, but multi-molecular combinations canhave this characteristic. In biochemistry, quantum dots are called redoxgroups. In nanotechnology, they are called quantum bits or qubits.Quantum dots typically have dimensions measured in nanometers, where onenanometer is 10⁻⁹ meter or a millionth of a millimeter. The fields ofbiology, chemistry, computer science, and electronics are all ofinterest to researchers in nanotechnology. An example of the overlappingof these disciplines is a hypothetical biochip, which might contain asophisticated computer and be grown in a manner similar to the way atree evolves from a seed. In this scenario, the terms redox group andqubit are equally applicable; it is hard to classify such a chip aseither animate or inanimate. The quantum dots in a biochip would eachaccount for at least one data bit, and possibly several.

The term “complementary” refers to the topological compatibility ormatching together of interacting surfaces of a ligand molecule and itsreceptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

The term “oxidation” means losing electron to oxidize. The term“reduction” means gaining electrons to reduce. The term “redox reaction”refers to any chemical reaction which involves oxidation and reduction.

The term “wafer” means a semiconductor substrate. A wafer could befashioned into various sizes and shapes. It could be used as a substratefor a microchip. The substrate could be overlaid or embedded withcircuitry, for example, a pad, via, an interconnect or a scribe line.The circuitry of the wafer could also serve several purpose, forexample, as microprocessors, memory storage, and/or communicationcapabilities. The circuitry can be controlled by the microprocessor onthe wafer itself or controlled by a device external to the wafer.

A “scribe line” is typically an “inactive” area between the active diesthat provide area for separating the die (usually with a saw). Often,metrology and alignment features populate this area.

A “via” refers to a hole etched in the interlayer of a dielectric whichis then filled with an electrically conductive material, preferablytungsten, to provide vertical electrical connection between stacked upinterconnect metal lines that are capable of conducting electricity.

“Metal lines” within a die are interconnect lines. Metal interconnectlines do not typically cross the scribe line boundary to electricallyconnect two dies or, as in the some embodiments of this invention, amultitude of die to one or more wafer pads.

The term “field effect transistor” (FET) is a family of transistors thatrely on an electric field to control the conductivity of a “channel” ina semiconductor material. FETs, like all transistors, can be thought ofas voltage-controlled resistors. Most FETs are made using conventionalbulk semiconductor processing techniques, using the single-crystalsemiconductor wafer as the active region, or channel.

The term “CMOS” means complementary metal oxide semiconductor.

“Micro-Electro-Mechanical Systems (MEMS)” include the integration ofmechanical elements, sensors, actuators, and electronics on a commonsilicon substrate through microfabrication technology. MEMS oftencombine electrical and mechanical functionalities on a single substrate.An example of a MEMS device could be a small mechanical chamber wheretwo liquids (biofluids, drugs, chemicals etc.) are mixed and a sensorinterprets the results. MEMS could also be integrated with logicfunctionalities i.e. having a CMOS circuit to perform some algorithmwith the data provided by the sensor. The CMOS circuit could then havecircuit elements that transport the results of the algorithm and thesensor input to another device (i.e. output to further devicescomprising the overall micro-system). While the electronics arefabricated using integrated circuit (IC) process sequences (e.g., CMOS,Bipolar, or BICMOS processes), the micromechanical components could befabricated using compatible “micromachining” processes that selectivelyetch away parts of the silicon wafer or add new structural layers toform the mechanical and electromechanical devices. Microelectronicintegrated circuits can be thought of as the “brains” of a system andMEMS augments this decision-making capability with “eyes” and “arms”, toallow microsystems to sense and control the environment. Sensors gatherinformation from the environment through measuring mechanical, thermal,biological, chemical, optical, and magnetic phenomena. The electronicsthen process the information derived from the sensors and through somedecision making capability direct the actuators to respond by moving,positioning, regulating, pumping, and filtering, thereby controlling theenvironment for some desired outcome or purpose. Because MEMS devicesare generally manufactured using batch fabrication techniques similar tothose used for integrated circuits, unprecedented levels offunctionality, reliability, and sophistication can be placed on a smallsilicon chip at a relatively low cost.

One of the mechanical processes typically performed by MEMS istransporting small amounts of fluids through channels, which are called“microfluidic channels.” These channels are frequently embedded in acovering layer (hereafter called: embedding layer). One example of amicrofluidic channel used in MEMS is in an electrokinetic pump.Electrokinetic pumps use an ionic fluid and a current imposed at one endof the channel and collected at the other end of the channel. Thiscurrent in the ionic fluid impels the ionic fluid towards the collectionpad of the electrokinetic pump.

The term “waveguide” refers to a device that controls the propagation ofan electromagnetic wave so that the wave is forced to follow a pathdefined by the physical structure of the guide. Generally speaking, theelectric and magnetic fields of an electromagnetic wave have a number ofpossible arrangements when the wave is traveling through a waveguide.Each of these arrangements is known as a mode of propagation. Opticalwaveguides are used at optical frequencies. An “optical waveguide” isany structure having the ability to guide optical energy. Opticalwaveguides may be (a) thin-film deposits used in integrated opticalcircuits (IOCs) or (b) optical fibers.

“Microprocessor” is a processor on an integrated circuit (IC) chip. Theprocessor may be one or more processor on one or more IC chip. The chipis typically a silicon chip with thousands of electronic components thatserves as a central processing unit (CPU) of a computer or a computingdevice.

One embodiment of the invention relates to a complex comprising a firstbinding partner, a first reporter associated with the first bindingpartner, an analyte, a second binding partner, and a second reporterassociated with the second binding partner. Preferably, the complexcomprises the first reporter, the first binding partner, the analyte,the second binding partner, and the second reporter in this order.Preferably, the first reporter or the second reporter comprises ananomaterial having more than 50%, 60%, 70%, 80% or 90% by weight ofinorganic content. The analyte and the binding partners preferablycomprise a biomolecule such as antibody, a protein, a carbohydrate, alipid, an antigen, a receptor, or a ligand, or a macromolecule. Thereceptor preferably comprises a quantum dot, a Raman tag, a fluorescenttag, a composite-organic-inorganic nanoparticle (COIN) or a magneticCOIN. The magnetic COIN could comprise a metal particle with at leastone Raman active organic compound adsorbed on the metal particle. In onevariation, the first reporter and the second reporter are associated tothe first binding partner and the second binding partner, respectively,through a molecule in between the first or second reporter and the firstor second binding partner.

Another embodiment of the invention relates to a device for analysiscomprising a microfluidic channel (MFC) comprising a plurality of firstbinding partners immobilized on spots in the MFC, wherein the MFCcomprises an inorganic support and an optically transparent cover andfurther comprises a plurality of probes (i.e., binding partners)optionally with COINs or magnetic COINs immobilized on spots in the MFC.The binding partner immobilized on the spot could be attached to ananalyte, which in turn could be attached another binding partnerattached to the analyte and a reporter such as a COIN or amagnetic-COIN.

The device for fluidic separation can be made by using soft lithographymethod with poly-dimethyl siloxane. With these techniques it is possibleto generate patterns with critical dimensions as small as 30 nm. Thesetechniques use transparent, elastomeric polydimethylsiloxane (PDMS)“stamps” with patterned relief on the surface to generate features. Thestamps can be prepared by casting prepolymers against masters patternedby conventional lithographic techniques, as well as against othermasters of interest. Several different techniques are known collectivelyas soft lithography. They are as described below:

Near-Field Phase Shift Lithography. A transparent PDMS phase mask withrelief on its surface is placed in conformal contact with a layer ofphotoresist. Light passing through the stamp is modulated in thenear-field. If the relief on the surface of the stamp shifts the phaseof light by an odd multiple of (, a node in the intensity is produced.Features with dimensions between 40 and 100 nm are produced inphotoresist at each phase edge.

Replica Molding. A PDMS stamp is cast against a conventionally patternedmaster. Polyurethane is then molded against the secondary PDMS master.In this way, multiple copies can be made without damaging the originalmaster. The technique can replicate features as small as 30 nm.

Micromolding in Capillaries (MIMIC). Continuous channels are formed whena PDMS stamp is brought into conformal contact with a solid substrate.Capillary action fills the channels with a polymer precursor. Thepolymer is cured and the stamp is removed. MIMIC is able to generatefeatures down to 1 μm in size.

Microtransfer Molding ((TM). A PDMS stamp is filled with a prepolymer orceramic precursor and placed on a substrate. The material is cured andthe stamp is removed. The technique generates features as small as 250nm and is able to generate multilayer systems.

Solvent-assisted Microcontact Molding (SAMIM). A small amount of solventis spread on a patterned PDMS stamp and the stamp is placed on apolymer, such as photoresist. The solvent swells the polymer and causesit to expand to fill the surface relief of the stamp. Features as smallas 60 nm have been produced.

Microcontact Printing ((CP). An “ink” of alkanethiols is spread on apatterned PDMS stamp. The stamp is then brought into contact with thesubstrate, which can range from coinage metals to oxide layers. Thethiol ink is transferred to the substrate where it forms aself-assembled monolayer that can act as a resist against etching.Features as small as 300 nm have been made in this way.

Techniques used in other groups include micromachining of silicon formicroelectricalmechanical systems (MEMS), and embossing of thermoplasticwith patterned quartz. Unlike conventional lithography, these techniquesare able to generate features on both curved and reflective substratesand rapidly pattern large areas. A variety of materials could bepatterned using the above techniques, including metals and polymers. Themethods complement and extend existing nanolithographic techniques andprovide new routes to high-quality patterns and structures with featuresizes of about 30 nm.

Applications of soft lithography in the near future could include simpleoptical devices, such as polarizers, filters, wire grids, and surfaceacoustic wave (SAW) devices. Longer term goals include working towardoptical data storage systems, flat panel displays, and quantum devices.Soft lithographic techniques are currently not competitive withconventional photolithography for multilayer fabrication where there arecritical requirements for pattern regularity.

Standard lithography on silicone wafer or silica glass could also beused to fabricate the devices of the embodiments of this invention.Chambers or channels can be make from the devices, fluidic flow can becontrolled by pressure gradient, electrical field gradient, gravity,heat gradient etc. The binding complexes can also be separated by planardevice with a single a plurality of chambers, where the surfaces aremodified with polymers (polyethylene glycol (PEG)-dramatized compounds)that can minimize non-specific binding. The substrate (solid support)can be inorganic material (e.g., glass, ceramic) or metal (e.g.,aluminum), biomolecules, protein, antibody, nucleic acid can be coatedon the surface for specific analyte binding.

The above embodiments could be practiced by the following methods.

Two-reporter method as shown in FIG. 1A: The complexes are formed whenan analyte is bound to two affinity binding partners each of which isassociated with a reporter. The reporter can be COIN(composite-organic-inorganic nanoparticles), quantum dots and otherRaman or fluorescent tags, but COINs will be particularly useful forthis purpose. Since many different types of COINs can be made and can beused to conjugate specific antibodies, a large collection of bindingcomplexes can be formed (reporter1-binding partner 1-analyte-bindingpartner 2-reportner 2). Preferably, reporter I is different fromreporter 2, which would allow the binding complex formed to bedistinguished over a molecule where reporter 1 or reporter 2 is notbound to the analyte. Unbound reporter-binding partners can be separatedby size or magnetic property if one of the reporters is paramagnetic.Binding partners can be antibodies, antigens, receptors or ligands.Sub-set of binding complexes is formed by controlling the concentrationof the complexes in solution. When multiple binding complexes movethrough a microfluidic channel, their optical features can be recordedand subsequently analyzed. Positive detection of an analyte is indicatedby the detection of two reporters simultaneously as predicted by bindingpartner specificity.

Spatial position and reporter method as shown in FIG. 1B: A devicesurface (array) containing multiple binding areas, each of the areascontains a mixture of binding partners (antibodies). The binding areascan be grouped into different compartments or spots, wherein one or moreof the compartments or spots could have an organic polymer layercontaining a polymeric brush or a linker molecule. Each of the bindingarea is optionally surrounded by non-binding surface and containsmultiple binding partners (different antibodies are mixed andimmobilized). The binding surfaces are fabricated to minimizenon-specific binding of analytes or reporters or binding partners. Abiological sample can be applied individually to a binding surface(area) or sub-set of the binding surfaces of the array. Thus multiplereporters can be located in a single binding area when sandwich bindingcomplexes are formed.

Another embodiment of the invention relates to a device for datacollection comprising a beam emitter, a MFC, a spectrometer,particularly a Raman spectrometer, and a detector. The beam emitter isto emit a beam comprising laser. The device could further comprise anoptical waveguide between the beam emitter and the MFC. As shown in FIG.1A, for example, the MFC could comprise a detection site to illuminate asample comprising a reporter attached to an analyte by the beam.Preferably, the spectrometer is a waveguide based spectrometer to createa phase shift in a Raman signal emitted by the sample. The detector isto detect a characteristic of the Raman signal emitted by the sample.The device could further comprise a microprocessor comprising softwareor a hardware to identify the Raman signal. The MFC could furthercomprise a plurality of first binding partners immobilized on spots inthe MFC. The MFC could further comprise a plurality of COINs or magneticCOINs immobilized on spots. The MFC could further comprise a microcoilor a Micro-Electro-Mechanical System (MEMS) device.

The devices of FIGS. 3 and 5 could be fabricated in the following way inone of the embodiments of the devices. The Fourier transformspectrometer can be fabricated using common semi-conductor fabricationtechniques. As an example a silicon wafer could be used as the startingmaterial. An oxide layer could be grown to be used as the bottomcladding of the waveguide. SiON could then be deposited to be used asthe waveguide core. This could then have waveguides patterned onto itusing wet or dry chemical etching. Control of the index of the MZI armscould be done using the thermo-optic effect by preferentially heatingthe MZI arms with heaters deposited onto the waveguide. Filters could beintegrated onto the waveguide by etching the upper surface or sidewallof the waveguide, or by varying the refractive-index of the waveguide asa function of position. An integrated photo-detector could be formed byfabricated a silicon PIN diode on the same substrate as the spectrometerin a way similar to obtaining planar optical devices that is known topersons of ordinary skill in this art.

The method steps and device for the above embodiment of the inventionare shown in FIGS. 2 and 3. Even though FIGS. 2 and 3 state“Raman-on-chip,” the embodiments of FIGS. 2 and 3 are equally applicableto other analyzers. In particular, the steps are as follows:

1. Mix a solution containing labeled probe molecules and labeled capturemolecules with the sample to be analyzed. Both the probe and capturemolecules have a COIN label. The sample could be a polymer, ananomaterial, a carbon nanotube, a nucleotide, or a biomaterial such apeptide, a protein, a ligand, a receptor, a sequence, DNA, RNA, etc.

2. Form a complex, which might involve hybridization, of the COINlabeled capture molecule, a target molecule of the sample and the COINlabeled probe molecule.

3. Detect the complex by simultaneous detection of two COIN labelsattached to the complex, which contains the first and second COIN labelsof the probe molecule and the target molecule, respectively.

The detection methodology is shown schematically in FIG. 3. The laserlight is focused into an optical waveguide, and is delivered to thesample which flows through a micro-fluid channel. The sample scatterslight and emits radiation, including Raman emission. The radiation fromthe sample would be collected by an optical waveguide basedspectrometer.

Another embodiment of the invention relates to a device for datacollection comprising a beam emitter, a chamber to hold a microarray, aspectrometer and a detector. The beam emitter is to emit a beamcomprising laser. The device could further comprise an optical waveguidebetween the beam emitter and the chamber. The chamber could comprise anoptical switch to detect the beam transmitted through the microarray.The spectrometer is preferably a waveguide based spectrometer to createa phase shift in a Raman signal emitted by the sample. The detector isto detect a characteristic of the Raman signal emitted by the sample.The device could further comprise a microprocessor comprising softwareor a hardware to identify the Raman signal. The device could furthercomprise a microarray, wherein the microarray comprises a plurality offirst binding partners immobilized on spots on the microarray. Themicroarray could further comprise a plurality of COINs or magnetic COINsimmobilized on spots.

The method steps and device of this embodiment of the invention areshown in FIGS. 4 and 5. Even though FIGS. 4 and 5 state “Raman-on-chip,”the embodiments of FIGS. 4 and 5 are equally applicable to otheranalyzers. In particular, the steps are as follows:

1. Introduce the sample and the COIN labeled probe molecules on asubstrate of a microarray having spots containing capture molecules(which may or may not be labeled). The sample could be a polymer, ananomaterial, a carbon nanotube, a nucleotide, or a biomaterial such apeptide, a protein, a ligand, a receptor, a sequence, DNA, RNA, etc.

2. Form a complex, which might involve hybridization, of the capturemolecule, a target molecule of the sample and the COIN labeled probemolecule.

3. Detect the complex by detection of one COIN label (or simultaneousdetection of two COIN labels if the capture molecule is a COIN labeledcapture molecule) attached to the complex.

The detection methodology to detect the sample on the microarray isshown schematically in FIG. 5. The laser light through an opticalwaveguide is focused on the microarray and the complex on microarraycould be illuminated from either above or below the microarray. Thecomplex emits its own signature spectrum comprising a Raman signal. Thesignature spectrum is collected by an optical waveguide basedspectrometer.

In the embodiments of the invention such as the two embodimentsdescribed above with reference to FIGS. 2-5, the sample receives thelaser light, and emits a unique spectrum of light specific to the COIN.A miniaturized spectrometer and detector could be placed to analyze thespectrum of the emitted light by a miniaturized spectrometer anddetector system, for example.

The above embodiments relating to FIGS. 1-5 could further include asample collection device for collecting the sample that has to beanalyzed by the analyzer of the embodiments of the invention. The samplecollection device could include suction and sample concentrationdevices. For example, a solid, liquid or gaseous sample could be suckedinto a sample collection device that produces a known background signal.Then, the sample could be concentrated within the sample collectiondevice. For example, a gas could be cooled to create condensate in thesample collection device. By concentrating the sample in the samplecollection device, it could reduce the analysis time, particularly for agaseous sample.

Another embodiment of the invention relates to a device for analyteconcentration comprising a first chamber comprising an analyte, amagnetic COIN, and a non-analyte, and a second chamber to hold aconcentrate comprising the analyte bound to the magnetic COIN.Preferably, the first chamber is exposed to a first magnetic field andthe second chamber is exposed to a second magnetic field, wherein thesecond magnetic field is different than the first magnetic field. Thefirst chamber could further comprise a magnet to produce the firstmagnetic field and the second chamber could further comprise a magnet toproduce the second magnetic field. In one variation of the device, thefirst chamber could comprise an instrument to drain a liquid comprisingthe non-analyte and the second magnetic field is the magnetic field ofthe earth.

In FIG. 6, the sample and the binding partner-conjugated magnetic COINsare mixed in a reaction chambers (centrifugation tubes), after thebinding, the binding complexes are cleaned by repeating washing withbuffers (applying magnetic field to concentrate and releasing the fieldto re-suspend the binding complexes). The cleaned binding complexes arethen transferred to a binding partner array chip (antibody chip, DNAchip, microfluidic chip etc., for example, see devices of FIG. 1).

The above embodiment could be practiced by the following method anddevice of FIG. 6:

Magnetic COIN as reporter and analyte carrier as shown in FIG. 6: Thedevice of FIG. 6 allows concentration in magnetic field of certainanalytes that are attached to magnetic COINs while excludingnon-analytes and other analytes that are not attached to the magneticCOINs. Magnetic COINs can be used as reporters as well as analytedetector and carriers. Analytes in solution can be captured by magneticCOINs that are conjugated with an affinity probe, and concentrated inmagnetic field. The concentrated sample with most non-analyte materialremoved can be placed on an affinity binding surface (with mixedaffinity binding partners (Abs)), thus different types sandwich bindingcomplexes can be formed with magnetic COINs as the reporters. The COINsignals can be detected by a Raman spectroscope. Each of the Ramanspectra can cover multiple COINs; multiple spectra are needed to collectsufficient data for analysis.

Yet another embodiment of the invention relates to an analyte separationmethod comprising capturing a first analyte in a solution by a magneticCOIN attached to a first binding partner to form a magneticCOIN-containing complex comprising the first analyte and the magneticCOIN attached to the first binding partner, capturing a second analytein the solution by a non-magnetic COIN attached to a second bindingpartner to form a non-magnetic COIN-containing complex comprising thesecond analyte and the non-magnetic COIN attached to the second bindingpartner, and grouping the magnetic COIN-containing complex separate fromthe non-magnetic COIN-containing complex on a surface of a microcoil bygenerating a local magnetic field on the surface of the microcoil. Themethod could further comprise controlling the current passing though themicrocoil to control a strength of the local magnetic field on thesurface of the microcoil. The method could further comprise releasingthe magnetic COIN-containing complex from the surface of the microcoil.

The method of manufacturing a microcoil of an embodiment of the deviceshown in FIG. 7 could be as follows. Briefly, dielectric material, whichcould be silicon oxide, silicon nitride, or a polymer material, such asbenzocyclobutene (BCB) dielectric layer, could be spun on a siliconwafer and cured. This BCB layer defines the separation distance betweenthe coils and the substrate. By using a sputter/lift-off process, thefirst Cu layer of 3 μm could be deposited and patterned. A second BCBlayer of 5 μm could be spun as an inter-layer-dielectric, which alsoplannerized the surface. Via holes could be opened and a second Cu layerof 3 μm deposited and patterned similarly. The coils could be passivatedby using a BCB layer on the top, which could be patterned to expose theprobing pads. The width of the Cu trace could range from 12 to 30 μm,the spacing was 12 μm.

The above embodiment could be practiced by the following method.

Microcoil for concentrating magnetic-COIN sandwich complexes shown inFIG. 7: Similar to FIG. 6, magnetic beads conjugated with a 1^(st) setof affinity binding partners (Abs) can be used to capture analytes insolution; non-magnetic COINs conjugated with a 2^(nd) set of affinitybinding partners are also in contact with the sample. Sandwich bindingcomplexes are formed, which can be sub-grouped on to microcoil surfaceswhen electricity is applied to generate local magnetic fields. Themicrocoil magnetic device can be fabricated using lithographytechniques. An electronic control board is used to control the currentpassing the microcoils and thus control the magnetic fields. The bindingcomplexes can be released and new sample can be introduced.

Yet another embodiment of the invention relates to a computerimplemented system comprising a first algorithm to simulate spectralfeatures produced by a hypothetical composition comprising a pluralityof reporters mixed in different ratios, a second algorithm to comparethe simulated spectral features with experimentally obtained spectralfeatures produced by an actual composition comprising a plurality ofreporters in different ratios, and a third algorithm to determine agoodness-of-fit between the simulated spectral features and theexperimentally obtained spectral features and to iteratively adjust thesimulated spectral features by adjusting the hypothetical composition tomaximize the goodness-of-fit to meet a pre-set statistical criteria. Itis possible that the first, second and third algorithms are bundled intoone or more software programs or one or more hardware components.Preferably, the plurality of reporters in the actual composition isassociated with a plurality of analytes of a biological sample. In onevariation, the goodness-of-fit is maximized by minimizing the differencebetween the between the simulated spectral features and theexperimentally obtained spectral features. Preferably, the differencebetween the between the simulated spectral features and theexperimentally obtained spectral features is determined by a geneticalgorithm that qualitatively optimizes the genetic algorithm, by aneural network that optimizes a set of selected parameters for aselected neural patterns or circuits, or by a principal componentanalysis that statistically decomposes components with maximumlikelihood.

For the device of FIG. 8, any device with low Raman background can beused, for example, solution on an aluminum surface, enclosed glasschambers, or any device described above for Raman measurement.

The above embodiment could be practiced by the following method anddevice of FIG. 8:

Machine learning and analyte quantification: FIG. 8 shows a generalscheme of machine learning (software training). Machine learning is apart of multiplex data analysis. When multiple reporters (COINs orquantum dots) are to be used in an assay, spectra of each of thesereporters at various concentrations are recorded. In a simplified dataanalysis a spectral peak (with a unique wavelength or wavenumber) andassociated combined parameters such as ratios or polynomials can be usedto quickly identify a single reporter. Computer simulation can be usedto predict the complex spectral features when a given set of reportersare mixed in a different ratios. The predications will be statisticallyverified by comparing the computations with actual experiments usingpre-determined/known compositions of reporters. The software algorithmand associated parameters are iteratively adjusted to maximize thegoodness-of-fit until the resulting converged algorithm and theassociated parameters meet pre-set statistical criteria. For example, avariety of algorithms and associated parameters can be used bystatistically adjusting and minimizing the energy of goodness-of-fitlandscapes such as genetic algorithm (focusing on qualitativelyoptimizing the algorithm), neural network (preset or empirical algorithmfocusing on optimizing a set of selected parameters for a selectedneural patterns/circuits), and principal component analysis (statisticaldecompositions of components with maximum likelihood) could be used. FFTdeconvolution also could be used to create a set of initial conditionsfor the said methods if necessary.

Analyte quantification: specific signal activity of a reporter needs tobe known before being used for an assay (single intensity per unitamount of reporter particles or molecules). For surface binding, onereporter can be considered to represent one analyte. In solutionbinding, the relation between reporter and analyte needs to bedetermined experimentally. When a spectrum is collected, trainedsoftware as described previously is used to deconvolute the spectrum todetermine the reporter concentration quantitatively. Based on reporters'specific activity and sample volume (surface area), multiple analytescan be quantified in a single assay.

FIG. 9 shows the result of multiplexing seven COIN Tags. From bottom tothe top, each plot in different color represents multiplexed COINmixtures, starting from two COINs successively up to seven COINs in asame solution. In brief, each addition of new COIN introduces uniquelyidentifiable peaks on top of the existing peaks. General “fingerprinting” method such as the machine learning software with statisticalsoftware described above could be effectively used to identify anddecompose the different signature components superimposed in the spectraof multiplex assay.

The embodiments of the invention could use silicon technology tofabricate interconnects for silicon chips to enable on-die synthesis ofpolymers such as DNA, peptides, and DNA-functionalized complementarynucleotide. Optionally, the embodiments of the invention could use waferprocessing cluster tools (process instruments) for synthesis. Typically,in volume silicon processing, a manufacturing line has a cluster ofinstruments (several identical instruments). Each can support a processstep or multiple process steps. By the embodiments of the invention,polymer synthesis can be treated as another process step in a devicemanufacturing line. A cluster of instruments can be configured within afacility to perform wafer level synthesis for efficient high volumemanufacturing.

The devices of the embodiments of the invention may be formed by anysuitable means of manufacture, including semiconductor manufacturingmethods, microforming processes, molding methods, material depositionmethods, etc., or any suitable combination of such methods. In certainembodiments one or more of the electrodes and/or the pad may be formedvia semiconductor manufacturing methods on a semiconductor substrate.Thin film inorganic coatings may be selectively deposited on portions ofthe substrate and/or pad surface. Examples of suitable depositiontechniques include vacuum sputtering, electron beam deposition, solutiondeposition, and chemical vapor deposition. The inorganic coatings mayperform a variety of functions. For example, the coatings may be used toincrease the hydrophilicity of a surface or to improve high temperatureproperties. Conductive coatings may be used to form electrodes. Coatingsmay be used to provide a physical barrier on the surface, e.g. to retainfluid at specific sites on the surface. The devices used in the presentinvention may be fabricated according to procedures well-known in thearts of microarray and semiconductor device manufacturing.

In some embodiments the probes may be selected from biomolecules, suchas polypeptides, polynucleotides, glycoproteins, polysaccharides,hormones, growth factors, peptidoglycans, or the like. The probe couldbe natural nucleotides such as ribonucleotides and deoxyribonucleotidesand their derivatives although unnatural nucleotide mimetics such as2′-modified nucleosides, peptide nucleic acids and oligomeric nucleosidephosphonates are also used. In embodiments employing oligonucleotideprobes, the probes may be synthesized, in situ, on the surface of thepad in either the 3′ to 5′ or 5′ to 3′ direction using the3′-β-cyanoethyl-phosphoramidites or 5′-β-cyanoethyl-phosphoramidites andrelated chemistries known in the art. In situ synthesis of theoligonucleotides may also be performed in the 5′ to 3′ direction usingnucleotide coupling chemistries that utilize 3′-photoremovableprotecting groups. Alternatively, the oligonucleotide probes may besynthesized on the standard controlled pore glass (CPG) in the 3′ to 5′direction using 3′-p-cyanoethyl-phosphoramidites and related chemistriesand incorporating a primary amine or thiol functional group onto the 5′terminus of the oligonucleotide. The oligonucleotides may then becovalently attached to the pad surface via their 5′ termini using thiolor amine-dependent coupling chemistries known in the art. The density ofthe probes on the surface can range from about 1,000 to 200,000 probemolecules per square micron. The probe density can be controlled byadjusting the density of the reactive groups on the surface of the padfor either the in situ synthesis or post-synthesis deposition methods.Other suitable means for synthesis of probe as are known in the art maybe employed.

The oligonucleotide probes include, but are not limited to, the fournatural deoxyribonucleotides; deoxythymidylic acid, deoxycytidylic acid,deoxyadenylic acid and deoxyguanylic acid. The probes can also beribonucleotides, uridylic acid, cytidylic acid, adenylic acid, andguanylic acid. Modified nucleosides may also be incorporated into theoligonucleotide probes. These include but are not limited to;2′-deoxy-5-methylcytidine, 2′-deoxy-5-fluorocytidine,2′-deoxy-5-iodocytidine, 2′-deoxy-5-fluorouridine,2′-deoxy-5-iodo-uridine, 2′-O-methyl-5-fluorouridine,2′-deoxy-5-iodouridine, 2′-deoxy-5(1-propynyl)uridine,2′-O-methyl-5(1-propynyl)uridine, 2-thiothymidine, 4-thiothymidine,2′-deoxy-5(1-propynyl)cytidine, 2′-O-methyl-5(1-propynyl)cytidine,2′-O-methyladenosine, 2′-deoxy-2,6-diaminopurine,2′-O-methyl-2,6-diaminopurine, 2′-deoxy-7-deazadenosine,2′-deoxy-6methyladenosine, 2′-deoxy-8-oxoadenosine,2′-O-methylguanosine, 2′-deoxy-7-deazaguanosine,2′-deoxy-8-oxoguanosine, 2′-deoxyinosine or the like.

The polynucleotide probes can vary in length from a range of about 5 toabout 100 nucleotides, such as about 8 to about 80 nucleotides, such asabout 10 to about 60 nucleotides, and such as about 15 to about 50nucleotides. Longer polynucleotide probes are typically employed forapplications where the sample contains a high sequence-complexity targetmixture. Shorter polynucleotide probes are typically employed inapplications where single nucleotide discrimination, such as mutationdetection, is desired.

The target molecule could be a nucleic acid such as genomic DNA, genomicRNA, messenger RNA, ribosomal RNA or transfer RNA, an oligonucleotide orpolynucleotide of DNA or RNA generated by enzymatic process such as PCRor reverse transcription, or any synthetic DNA, RNA, or any otherdesired nucleic acid or any combination thereof. The target molecule maybe double stranded or single stranded. It is preferred that the targetmolecule be single stranded in order to increase the efficiency of itsinteraction with the probe sequences. The target molecule could containnanomaterials such a carbon nanotube, wherein the nanomaterial such asthe carbon nanotube could be functionalized at its ends to moleculescontaining nucleic acid.

The architecture of the array probes may be either generic or specificwith regard to the complementary target sequences that it may hybridizewith. For example, an array of all possible 7-mer probe sequences couldbe used to interrogate targets having any sequence. The advantage ofsuch an array is that it is not application specific and thereforegeneric. Alternatively, the probe array may contain polynucleotidesequences that are complementary to a specific target sequence or set oftarget sequences and individual or multiple mutations thereof. Such anarray is useful in the diagnosis of specific disorders, which arecharacterized by the presence of a particular nucleic acid sequence. Forexample, the target sequence may be that of a particular exogenousdisease causing agent, e.g. human immunodeficiency virus, oralternatively the target sequence may be that portion of the humangenome which is known to be mutated in instances of a particulardisorder, e.g., sickle cell anemia or cystic fibrosis, or to a portionof a genome known to be associated with certain phenotypes, e.g.,resistance to certain drugs, over-reactivity to certain drugs, or evensusceptibility to side-effects of certain drugs.

In one embodiment of the present invention, polymers on a plurality ofdies on a wafer substrate are functionalized on the electrodes asfollows. First, a terminal end of a monomer, nucleotide, or linkermolecule (i.e., a molecule which “links,” for example, a monomer ornucleotide to a substrate) is provided with at least one reactivefunctional group, which is protected with a protecting group removableby an electrochemically generated reagent. The protecting group(s) isexposed to reagents electrochemically generated at the electrode andremoved from the monomer, nucleotide or linker molecule in a firstselected region to expose a reactive functional group. The substrate isthen contacted with the monomer or a pre-formed molecule (called thefirst molecule) such that the surface bonds with the exposed functionalgroup(s) of the monomer or the pre-formed molecule. The first moleculemay also bear at least one protected chemical functional group removableby an electrochemically generated reagent. The monomer or pre-formedmolecule can then be deprotected in the same manner to yield a secondreactive chemical functional group. A different monomer or pre-formedmolecule (called the second molecule), which may also bear at least oneprotecting group removable by an electrochemically generated reagent, issubsequently brought in the vicinity of the substrate to bond with thesecond exposed functional group of the first molecule. Any unreactedfunctional group can optionally be capped at any point during thesynthesis process. The deprotection and bonding steps can be repeatedsequentially at the plurality of the predefined regions on the substrateuntil polymers or oligonucleotides of a desired sequence and length areobtained.

In another embodiment of the present invention, polymers on a pluralityof dies on a wafer substrate are functionalized on the electrodes asfollows. First, a substrate of a wafer having one or more moleculesbearing at least one protected chemical functional group bonded on anarray of electrodes on a plurality of dies is obtained. The array ofelectrodes is contacted with a buffering or scavenging solution.Following application of an electric potential to selected electrodes inthe array of electrodes sufficient to generate electrochemical reagentscapable of deprotecting the protected chemical functional groups,molecules on the array of electrodes are deprotected to expose reactivefunctional groups, thereby preparing them for bonding. A monomersolution or a pre-formed molecule (called the first molecule), such asproteins, nucleic acids, polysaccharides, and porphyrins, is thencontacted with the substrate surface of the wafer and the monomers orpre-formed molecules are bonded in parallel with a plurality ofdeprotected chemical functional groups on a plurality of dies on thewafer. Another sufficient potential is subsequently applied to selectelectrodes in the array to deprotect at least one chemical functionalgroup on the bonded molecule or another of the molecules bearing atleast one protected chemical functional group on a plurality of dies onthe wafer. A different monomer or pre-formed molecule (called the secondmolecule) having at least one protected chemical functional group issubsequently attached to a deprotected chemical functional group of thebonded molecule or the other deprotected molecule located at a pluralityof dies of the wafer. The selective deprotection and bonding steps canbe repeated sequentially until polymers or oligonucleotides of a desiredsequence and length are obtained. The selective deprotection step isrepeated by applying another potential sufficient to effect deprotectionof a chemical functional group on a bonded protected monomer or a bondedprotected molecule. The subsequent bonding of an additional monomer orpre-formed molecule to the deprotected chemical functional group(s)until at least two separate polymers or oligonucleotides of desiredlength are formed on the substrate.

Some of the advantages of the embodiments of the invention include:

-   -   Fast data collection, e.g., in array-based assays, one spectrum        contain multiple data points; and in fluidic-based assays,        magnetic force-assisted detection concentrated the analyte-probe        complex, reducing scanning time)    -   High throughput (multiple tests can be performed at the same        time in multiplexed analysis).    -   Use of small sample volume (crucial to clinical diagnosis)    -   Low cost (Less samples, reagents, and labor).

This embodiment of the invention relate to generating multiplex data andanalyzing the resulting data. The embodiments of the invention can beused to collect information from multiple binding complexes in a singlemeasurement (1 data integration time, for example 0.1 second); normallya separation step is used before any detection (for example, magneticseparation, centrifugation, etc.). The embodiments of the invention aredifferent from current methods which uses beads, which are likely to bemuch larger than COINs and rely on a correlation between thefluorescence of the classification laser and the fluorescence of thereporter to detect a single type of binding complexes, while theCOIN-based assay of some of the embodiments of the invention does notneed this type of statistical correlation method because the label/tagsignatures of COINs are directly read by Raman system.

The embodiments of the invention can be used to carry out theelectrochemical syntheses of polymers such as DNA and peptides accordingto any of a variety of approaches known to person skilled in the art.For example, any of a variety of reduction/oxidation (redox) reactionsmay be employed to electrochemically control the localization and pH ofa solution on Si-based electrodes to enable the attachment andelongation of polymers. In such methods, the electrical current drivesthe oxidation of an appropriate molecule at the anode(s) and thereduction of another molecule at the cathode(s) to control the kineticsand stoichiometry of acid-catalyzed organic syntheses on a Si-basedcircuit Such methods can also be used to generate high pH (basic)solutions, and to drive any other electrochemical redox reactions knownto one skilled in the art that may or may not result in pH changes(e.g., can also be used to generate reactive free radicals).

Another embodiment of the invention is electrochemical detection usingthe array chip. Typically these methods employ measurements of currentflow across a DNA monolayer tethered to a circuit on a siliconsubstrate. Current flow properties proportionately change when the DNAmonolayers are bound by an appropriate redox molecule-tagged test DNA oruntagged DNA that is co-added with a redox-active molecule thatspecifically binds double stranded DNA. Enzyme amplification methods canalso be incorporated into such assays in order to enhance theelectrochemical signal generated by binding events. Note that thesemethods can also be adapted by one skilled in the art to measure thebinding between other molecular species such as between two proteins ora protein and a small molecule.

The array chip could also be used for therapeutic materials development,i.e., for drug development and for biomaterial studies, as well as forbiomedical research, analytical chemistry, high throughput compoundscreening, and bioprocess monitoring. An exemplary application includesapplications in which various known ligands for particular receptors canbe placed on the array chip and hybridization could be performed betweenthe ligands and labeled receptors.

Yet another application of the array chip of an embodiment of thisinvention includes, for example, sequencing genomic DNA by the techniqueof sequencing by hybridization. Non-biological applications are alsocontemplated, and include the production of organic materials withvarying levels of doping for use, for example, in semiconductor devices.Other examples of non-biological uses include anticorrosives,antifoulants, and paints.

It is specifically contemplated that the array chip and/or the methodsof manufacturing the array chip of an embodiment of the invention couldbe used for developing new materials, particularly nanomaterials formany purposes including, but not limited to corrosion resistance,battery energy storage, electroplating, low voltage phosphorescence,bone graft compatibility, resisting fouling by marine organisms,superconductivity, epitaxial lattice matching, or chemical catalysis.Materials for these or other utilities may be formed proximate to one ora plurality of the electrodes in parallel on a plurality of dies of asilicon wafer, for example. Alternatively, materials may be formed bymodifying the surface of one or a plurality of electrodes on a pluralityof dies by generating reagents electrochemically.

It is further contemplated that an array chip of the embodiments of theinvention could be used to develop screening methods for testingmaterials. That is, reagents electrochemically generated by an electrodeon a die could be used to test the physical and chemical properties ofmaterials proximate to the electrode. For example, the array chip couldbe used for testing corrosion resistance, electroplating efficiency,chemical kinetics, superconductivity, electro-chemiluminescence andcatalyst lifetimes.

The advantageous characteristics of some of the embodiments of theinvention are illustrated in the examples, which are intended to bemerely exemplary of the invention.

The array chips of the embodiments of the invention are preferablysilicon bio-chips built by using silicon process technology and SRAMlike architecture with circuitries including electrode arrays, decoders,serial-peripheral interface, on chip amplification, for example.

The embodiments of this invention have several practical uses. Forexample, one embodiment of the invention allows molecules andnanomaterials detection/analysis based on the electrical readout ofspecific binding events (target to functionalized electrodes withprobes) using CMOS-based devices. Another embodiment of the inventionhas potential applications for nanomaterials study (for example, in-situanalysis of DNA-mediated assembly of carbon nano-tubes on functionalizedelectrodes) to be used in electronic devices (CNT transistors andinterconnects) as well as well as for detection of bio-species (DNA,protein, viruses etc.) for molecular diagnostics, homeland security,drug discovery and life science R&D work. Yet another embodiment of theinvention could be to use Nanomaterials, such as carbon-nanotubes, inpotential applications as interconnect materials. Carbon-nanotubes havelower resistivity than Cu and higher electromigration resistance (1000×higher than Cu). Yet another application could be to develop DNAfunctionalized electrodes with CMOS circuitry for immobilizing,detection, addressing, electrical readout and amplification of thesignal can find potential application in silicon DNA chips. Siliconchips with DNA functionalized electrodes could find potentialapplication to build nano-structures and in-situ assembly study ofnanomaterials. Silicon DNA chips could also find potential applicationin medical diagnostics, homeland security devices, drug discovery andlife science R&D work.

This application discloses several numerical range limitations thatsupport any range within the disclosed numerical ranges even though aprecise range limitation is not stated verbatim in the specificationbecause the embodiments of the invention could be practiced throughoutthe disclosed numerical ranges. Finally, the entire disclosure of thepatents and publications referred in this application, if any, arehereby incorporated herein in entirety by reference.

1-40. (canceled)
 41. A computer implemented system comprising a firstalgorithm to simulate spectral features produced by a hypotheticalcomposition comprising a plurality of reporters mixed in differentratios, a second algorithm to compare the simulated spectral featureswith experimentally obtained spectral features produced by an actualcomposition comprising a plurality of reporters in different ratios, anda third algorithm to determine a goodness-of-fit between the simulatedspectral features and the experimentally obtained spectral features andto iteratively adjust the simulated spectral features by adjusting thehypothetical composition to maximize the goodness-of-fit to meet apre-set statistical criteria.
 42. The computer implemented system ofclaim 41, wherein the first, second and third algorithms are bundledinto one or more software programs or one or more hardware components.43. The computer implemented system of claim 41, wherein the pluralityof reporters in the actual composition is associated with a plurality ofanalytes of a biological sample.
 44. The computer implemented system ofclaim 41, wherein the goodness-of-fit is maximized by minimizing thedifference between the simulated spectral features and theexperimentally obtained spectral features.
 45. The computer implementedsystem of claim 44, wherein the difference between the between thesimulated spectral features and the experimentally obtained spectralfeatures is determined by a genetic algorithm that qualitativelyoptimizes the genetic algorithm, by a neural network that optimizes aset of selected parameters for a selected neural patterns or circuits,or by a principal component analysis that statistically decomposescomponents with maximum likelihood. 46-68. (canceled)
 69. A computerimplemented method comprising simulating spectral features product by ahypothetical composition comprising a plurality of reporters mixed indifferent ratios, comparing the stimulated spectral features withexperimentally obtained spectral features produced by an actualcomposition comprising a plurality of reporters in different ratios,determining a goodness-of-fit between the simulated spectral featuresand the experimentally obtained spectral features and iterativelyadjusting the stimulated spectral features by adjusting the hypotheticalcomposition to maximize the goodness-of-fit to meet a pre-setstatistical criteria.
 70. The computer implemented method of claim 69,wherein the goodness-of-fit is maximized by minimizing the differencebetween the simulated spectral features and the experimentally obtainedspectral features.